Dynamically-adaptive live therapeutic agents and methods of use thereof

ABSTRACT

This disclosure provides microbes engineered to detect virulent and spore states of pathogens and release an appropriate therapeutic response accordingly and compositions and methods of use of the same.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The United States Government has rights in this application pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory.

BACKGROUND

Current use, and the misuse or overuse, of broad spectrum antibiotics has significantly limited their effectiveness against pathogens, especially those pathogens that have life-cycles that can evade these drugs. As pathogens develop new resistance mechanisms, the ability to treat even common infectious diseases diminishes and results in higher medical expenses, prolonged illnesses, and even death.

Clostridium difficile (C. difficile or C. diff) bacterial infections cause diarrhea as well as more serious intestinal conditions such as colitis. C. difficile is a leading cause of intestinal infection and is common in people on prolonged antibiotic regimens, the elderly, and those in hospitals. C. difficile has proven difficult to treat, with recurrence resulting in more than 20% of patients. Recurrence is caused in part because C. difficile is naturally resistant to broad spectrum antibiotics due to formation of a dormant, spore state. Suppression of natural gut microbiota during antibiotic treatment further contributes to the spread and impact of C. difficile. In fact, C. difficile kills more than 14,000 people a year in the United States alone and adds approximately $4.8 billion in annual U.S. healthcare costs.

Novel approaches that can respond dynamically with the changing life cycle states of resistant pathogens are needed.

SUMMARY

Methods and materials are provided for detecting and responding to different pathogen states.

In some aspects the disclosure provides genetically engineered microbes comprising a dual pathogen state detection system, the dual pathogen detection system comprising: (a) a first sensor for detection of a virulent form of a pathogen, wherein the first sensor is operatively connected to a virulent secretion system; and (b) a second sensor for detection of a spore form of the pathogen, wherein the second sensor is operatively connected to a spore secretion system.

In some aspects the disclosure provides compositions comprising an amount of the genetically engineered microbe according to any embodiment disclosed and described herein.

In some aspects the disclosure provides methods of inhibiting a pathogenic microbe comprising delivering a genetically engineered microbe as in any embodiment disclosed and described herein to a composition comprising the pathogenic microbe.

In some aspects the disclosure provides methods of detecting a state of a pathogenic microbe comprising delivering the genetically engineered microbe as in any embodiment disclosed and described herein to a composition comprising the pathogenic microbe.

In some aspects the disclosure provides methods of treating or preventing a pathogenic infection in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising genetically engineered microbes as in any embodiment disclosed and described herein.

In some embodiments, the microbe is a bacterium, for example, a Gram-positive bacterium. In some embodiments, the bacterium is Escherichia coli Nissle 1917 or Lactococcus lactis (L. lactis). In some embodiments, the microbe is a viable microbe.

In some embodiments, at least the first sensor or the second sensor detects a protein on the surface of the pathogen.

In some embodiments, the modified cell surface receptor comprises an antibody, antimicrobial peptide, or fragment thereof.

In some embodiments, the first sensor detects a toxin secreted by the virulent pathogen, for example, TcdA or TcdB.

In some embodiments, the second sensor detects a cell surface protein on the pathogen.

In some embodiments, the cell surface protein is a cell wall protein or a spore coat protein, for example, a BclA glycoprotein or a cysteine (CdeC)-rich protein.

In some embodiments, at least one of the first sensor or the second sensor does not detect a quorum sensing molecule.

In some embodiments, the virulent secretion system produces one or more agents in an encapsulated shell, one or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (gRNA), or any combination thereof.

In some embodiments, the encapsulated agent is a bacteriocin or a modified derivative thereof. In some embodiments, the bacteriocin is selected from the group consisting of colicin, diffocin, pyocin, and rhuricin 17.

In some embodiments, the encapsulated agent is an autolysin, an endolysin, an antimicrobial peptide, an antitoxin, or any combination thereof.

In some embodiments, the encapsulated shell is modified with an antibody. In some embodiments, the antibody targets intestinal epithelial cells.

In some embodiments, the spore secretion system produces encapsulated agents. In some embodiments, the encapsulated agents induce germination of spores. In some embodiments, the encapsulated agents are bile salts. In some embodiments, the encapsulated agent is a protease.

In some embodiments, the microbe further comprises a microbe death trigger.

In some embodiments, the composition is a probiotic, a food, a nutraceutical, a pharmaceutical, a biospray and/or a beverage. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier/excipient.

In some embodiments, the pathogenic microbe is part of a population of cultured cells (i.e., in vitro). In some embodiments, the pathogenic microbe is part of a population of cells of a subject (i.e., in vivo).

In some embodiments, the state is a virulent state. In other embodiments, the state is a spore state.

In some embodiments, the pathogenic infection is a recurrent pathogenic infection.

In some embodiments, the pathogen infection is caused by an intestinal and/or gastrointestinal pathogen, for example, Clostridium difficile.

In some embodiments, the subject experiences and/or reports fewer or less severe side effects on natural gut microbiota as compared to a conventional therapy, for example, the conventional therapy is an antibiotic.

In some embodiments, the genetically engineered microbes are locally administered.

In some embodiments, the composition comprising genetically engineered microbes is administered sequentially or concurrently with an antibiotic. In some embodiments, the composition is administered after the antibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a schematic of the screening process of cell binding domains/antibodies, endolysins and antimicrobial peptides for developing engineered cell surface signaling receptors that have virulent binding domains and that initiate release of a CRISPR/phage therapy that target and kill pathogens.

FIG. 2 presents several schematics of modular pathogen state detection systems according to an embodiment of the present disclosure.

FIG. 3 presents a schematic of a therapeutic microbe according to an embodiment of the present disclosure, engineered to detect and respond to both virulent and spore forms of a pathogen and to calculate when and whether to undergo lysis to release a therapeutic agent.

FIG. 4 presents a schematic of a therapeutic agent according to an embodiment of the present disclosure, engineered to sense the changing state of a pathogen within a host, process the information using engineered signaling cascades, and deliver specific, scalable, and effective countermeasures against the sensed state. Upon detection of a spore coat protein on the surface of the dormant spore form, the engineered microbe releases a spore-targeting agent. Upon binding of a toxin (circles) released from the virulent form, the engineered microbe releases via lysis an encapsulated virulent targeting agent.

FIG. 5 presents a general schematic of a dynamically adaptive therapeutic agent according to an embodiment of the present disclosure, engineered to be able to detect and respond to both spore and virulent forms of C. difficile, as well as mitigate toxic effect of toxin on host tissues.

DETAILED DESCRIPTION

Conventional treatments for pathogenic infections often utilize broad spectrum antibiotics. Such antibiotics, like ampicillin for example, revolutionized medicine for their ability to act against a wide range of disease causing bacteria. However, these antibiotics indiscriminately target both the pathological bacteria and the natural, beneficial microbiota. Destruction of the body's natural bacteria provides an environment for drug resistant microbes to prosper and lead to secondary infection. One common secondary infection is C. diff. Importantly, use of “silver bullet” antibiotics as a single treatment regimen often does not meet all the requirements for efficacy, drug delivery, and low host toxicity. In particular, pathogens adapt and evolve to these static treatments such that the treatments become ineffective.

The present disclosure relates to the use of live therapeutic agents (i.e., genetically engineered microbes) that can dynamically sense and respond to the current state of a pathogen. Using multiple mechanisms of attack, these agents act, in part, to slow down the evolution of resistance. These therapeutic agents can be specifically tailored to identify and respond to a particular pathogen and can be locally released to minimize the effects on the host's tissue and natural microbiota.

After reading this description it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

The detailed description is divided into various sections only for the reader's convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present disclosure.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); McPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); McPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd) edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/−15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of cells.

Definitions

As used herein the following terms have the following meanings:

The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

“Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

As used herein the term “operatively connected,” such as in reference to a sensor for detection of a pathogen and a secretion system means the sensor and the secretion system are connected in such a way that they work together, for example, via cell signaling. The elements that are operatively connected do not need to actually touch, but one element acts on the other.

Microbes

Aspects of the disclosure provide genetically engineered microbes. Suitable microbes include, for example, bacteria (e.g., Lactobacillus), yeast (e.g., Saccharomyces and Candida), and algal. In some embodiments, the microbe is a bacterium, for example a Gram-positive or a Gram-negative bacterium.

Non-limiting examples of suitable bacteria include Acetobacter spp., Acidithiobacillus spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Arthrobacter spp., Azotobacter spp., Bacillus spp., Chromobacterium spp., Citrobacter spp., Clostridium spp., Comamonas spp., Corynebacterium spp., Escherichia spp., Flavobacterium spp., Geobacillus spp., Geobacter spp., Gluconobacter spp., Lactobacillus spp., Lactococcus spp., Microlunatus spp., Mycobacterium spp., Pantoea spp., Pseudomonas spp., Ralstonia spp., Rhizobium spp., Rhodococcus spp., Saccharopolyspora spp., Salmonella spp., Serratia spp,. Sinorhizobium spp., Stenotrophomonas spp., Streptococcus spp., Streptomyces spp., Synechocystis spp., Thermus spp., Xanthomonas spp., and Zymonas spp.

In some embodiments, the bacterium is a probiotic. Probiotics are live microorganisms, which when administered in adequate amounts confer a health benefit on the host. Non-limiting examples of probiotic bacteria include Escherichia coli Nissle 1917, Lactococcus Lactis, Lactobacillus Acidophilus, Lactobacillus Brevis, Lactobacillus Bulgaricus, Lactobacillus Casei, Lactobacillus Helveticus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus Rhamnosus GG, Lactobacillus rhamnosus GR-1, Lactobacillus plantarum, Lactobacillus Silivarius, Eubacterium hallii and Bifidobacterum Bifidum, Bifidobacterum Breve, Bifidobacterium Infantis, Bifidobacterium Lactis, Bifidobacterium longum, Bacillus Coagulans, Saccharomyces Boulardii, Streptococcus Thermophilus or a combination thereof. In one preferred embodiment, the bacteria is Lactococcus Lactis (L. lactis).

Non-limiting examples of suitable yeast include Brettanomyces spp., Candida spp., Debaryomyces spp., Kluyveromyces spp., Pachysolen spp., Paffia spp., Pichia spp., Saccharomyces spp., Schizosaccharmoyces spp., Talaromyces spp., and Yarrowia spp. In one embodiment, the yeast is a modified Saccharmoyces cerevisiae.

In some embodiments, the microbe is a viable microbe.

Engineered Microbes

Aspects of the disclosure encompass genetically engineered microbe comprising a pathogen state detection system, in particular a dual pathogen state detection system. The detection systems comprise a sensor on the surface of the microbe that detects the presence of a pathogen. Detection can occur directly (i.e., detection of a pathogen cell surface marker) or indirectly (i.e., detection of a signal produced by the pathogen (e.g., a toxin). In a preferred embodiment, the dual pathogen state detection system of the microbes comprise at least a first sensor for detection of a virulent form of a pathogen and a second sensor for detection of a spore form of the pathogen. The terms “state” and “form” can be used interchangeably in reference to a pathogen. For example, the spore state is equivalent to the spore form.

In some embodiments, at least the first sensor or the second sensor detects a protein on the surface of the pathogen. In other embodiments, the first sensor detects a protein on the surface of the pathogen and the second sensor detects a non-surface pathogen signal. In yet other embodiments, the second sensor detects a protein on the surface of the pathogen and the first sensor detects a non-surface pathogen signal. In still other embodiments, both the first sensor and the second sensor detect a protein on the surface of the pathogen.

In some embodiments, the first sensor, the second sensor, or both is a modified cell surface receptor. A cell surface receptor can be modified by any method known in the art. Non-limiting examples of modified cell surface receptors include antibodies, endolysins, and antimicrobial peptides, and fragments thereof. In some embodiments, the modified cell surface receptor comprises an antibody, antimicrobial peptide, or fragment thereof.

In one embodiment, the antibody or fragment thereof is a monoclonal antibody. In another embodiment, the antibody is a polyclonal. Suitable antibodies or fragments thereof for use with the present disclosure include, for example, actoxumab, bezlotoxumab, anthem, pagibaximab, efibazumab, urtoxazumab. As used herein the term “antibody” or “antibodies” refers to immunoglobulin molecules and immunologically active portions or immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immune-specifically bind an antigen). The term also refers to antibodies comprise of two immunoglobulin heavy chains and two immunoglobulin light chains as well as any variety of forms including full length antibodies and portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFV, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies (e.g., Lanzavechhia et al., (1987) Eur. J Immunol. 17, 105) and single chains (e.g., Huston et al., Proc. Natl. Acad Sci. U.S.A., (1988) 85, 5879-5883 and Bird et al., (1988) Science 242, 423-426, which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed. (1984); Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapiller and Hood, (1986) Nature, 323, 15-16, which are incorporated herein by reference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE or IgD). Preferably, the antibody is IgG. An antibody may be non-human (e.g., from mouse, goat, or any other animal), fully human, humanized, or chimeric.

Antimicrobial peptides (AMPs) are a component of the innate immune system and play a critical role in warding off invading pathogens. To date, more than 2,500 peptides have been isolated and characterized. Wang et al, (2004) Nucleic Acids Res. 32(Database issue): D590-2. Suitable AMPS include, for example, HBD1-3, HNP1, HD5 cathelicidins (e.g., LL-37), coprisin, polymixin, and nisin.

In one preferred embodiment, at least one of the first sensor or the second sensor does not detect a quorum sensing molecule. A quorum sensing molecule refers to a molecule produced by a cell that can signal population density of the population of cells containing that cell. In some embodiments, a quorum sensing molecule is an oligopeptide, an N-Acyl homoserine lactone (AHL), an autoinducer or a pheromone.

Virulent Detection and Secretion System

The disclosure provides a first sensor for detection of a virulent form of a pathogen, wherein the first sensor is operatively connected to a virulent secretion system.

In some embodiments, the first sensor detects a compound, for example a toxin, secreted from the pathogen. Bacterial generate toxins which are classified as exotoxins or endotoxins. Exotoxins are secreted, while endotoxins remain part of the bacteria. In some embodiments, the first sensor detects large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens, or Clostridium novyi. In some embodiments, the toxin is C. difficile toxin A (TcdA), toxin B (TcdB), or binary toxin A (CDTa). Toxin A is an enterotoxin that causes diarrhea. Toxin B is a cytotoxin that kills cells.

Other non-limiting examples of bacterial toxins include, Bordetella pertussis AC toxin (A/B) and Bacillus anthracis EF, Botulinum neurotoxin (BoNT), tetanus toxin (TeNT protein), staphylococcal toxin, alpha toxin, anthrax toxin, cholera toxin, cyanotoxin, diphtheria toxin, E. coli heat-labile toxin LT, pertussis toxin, Pseudomonas toxin A, shiga toxin, shiga-like toxin, and Staphylococcus aureus Exfoliatin B.

In some embodiments, the first sensor detects a cell surface protein on the surface of the virulent pathogen. In some embodiments, the cell surface protein is Cwp66 adhesin protein, S-layer proteins (e.g., HMW-SLP), or flagellin protein (e.g., FlaA, FlgE). In some embodiments, the cell surface protein is a spore coat proteins, for example, BclA glycoproteins or CdeC.

In some embodiments, the first sensor detects a catalytic or cell wall binding domain of C. difficile. In some embodiments, the catalytic or cell wall binding domain from C. difficile includes any one of SEQ ID NOS. 1-36 or a portion thereof.

In some embodiments, the first sensor detects a catalytic or cell wall binding domain of Clostridium phages. In some embodiments, the catalytic or cell wall binding domain from Clostridium phages includes any one of SEQ ID NOS. 37-40 or a portion thereof.

In one preferred embodiment, first sensor for detection of a virulent form of a pathogen does not detect a quorum sensing molecule.

The microbes of the present disclosure also provide a virulent secretion system which is operatively connected to the first sensor.

The virulent secretion system can produce one or more agents in an encapsulated shell, one or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (gRNA), or any combination thereof. An example of CRISPR targets include Frizzled proteins (as described in Tao et al. (2016) Nature 538:350-355. In some embodiments, the CRISPR target is a toxin gene, for example, TcdA or TcdB. In some embodiments, the encapsulated agent is a bacteriocin, such as, colicin, diffocin, pyocin, and rhuricin 17, or a modified derivative thereof. In other embodiments, the encapsulated agent is an autolysin, an endolysin, an antimicrobial peptide, an antitoxin, or any combination thereof.

The encapsulation shells can be created by any method known in the field, for example, by the method described by Moon et al. (2014) which is hereby incorporated by reference in its entirety. Moon et al. (2014) Biomacromolecules 15 (10), 3794-3801. The encapsulation shells can be modified by any known method. For example, the encapsulated shell is modified with an antibody. In one embodiment, the antibody targets intestinal epithelial cells.

Spore Detection and Secretion System

The disclosure also provides a second sensor for detection of a spore form of the pathogen, wherein the second sensor is operatively connected to a spore secretion system.

In some embodiments, the second sensor detects a cell surface protein on the spore pathogen, for example, a cell wall protein or a spore coat protein. Non-limiting examples of spore coat proteins include a BclA glycoprotein and a cysteine (CdeC)-rich protein.

In one preferred embodiment, the second sensor for detection of a spore form of a pathogen does not detect a quorum sensing molecule.

The microbes of the present disclosure also provide a spore secretion system which is operatively connected to the second sensor. In some embodiments, the spore secretion system produces encapsulated agents.

In some embodiments, wherein the encapsulated agents induce germination of spores, for example, bile salts. In other embodiments, the encapsulated agent is a protease.

In some embodiments, the microbes disclosed herein further comprise a microbe death trigger (e.g., a suicide cassette). Cells may contain suicide cassettes comprising inserted sequences encoding certain reporter proteins (e.g., thymidine kinase (TK)). In some embodiments, suicide cassettes are used to facilitate the identification of cells from a larger cell population. In other embodiments, suicide cassettes are used to destroy microbes which have proliferated to an undesirable level in vivo. Microbes can be further modified to comprise a tracking system to follow the in vivo position and ultimately final location and/or clearance of the microbes following introduction into a subject. For example, the engineered microbes may be modified to comprise a fluorescent detectable marker (e.g., green fluorescent protein (GFP)).

The microbes of the present disclosure can be genetically modified by any suitable methodology. As a non-limiting example, one or more of the nucleic acids (e.g., nucleic acid encoding for the first sensor, the second sensor, the virulent secretion system, and/or the spore secretion system) associated with the disclosure can be expressed in a recombinant expression vector. As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted, such as by restriction and ligation, for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.

A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which can be further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

An expression vector is one into which a desired nucleic acid sequence may be inserted, for example by restriction and ligation, such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined. When the nucleic acid molecule that encodes any of the genes associated with the claimed invention is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. A nucleic acid molecule that comprises a gene associated with the invention can be introduced into a cell or cells using methods and techniques that are standard in the art.

A nucleic acid, polypeptide or fragment thereof described herein can be synthetic. As used herein, the term “synthetic” means artificially prepared. A synthetic nucleic acid or polypeptide is a nucleic acid or polypeptide that is synthesized and is not a naturally produced nucleic acid or polypeptide molecule (e.g., not produced in an animal or organism). It will be understood that the sequence of a natural nucleic acid or polypeptide (e.g., an endogenous nucleic acid or polypeptide) may be identical to the sequence of a synthetic nucleic acid or polypeptide, but the latter will have been prepared using at least one synthetic step.

Compositions

The disclosure also provides compositions comprising an amount of the genetically engineered microbes disclosed and described herein. In some embodiments, the composition is a probiotic, a food, a nutraceutical, a pharmaceutical, a biospray and/or a beverage.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier/excipient. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The composition may comprise a pharmaceutically acceptable excipient, a pharmaceutically acceptable salt, diluents, carriers, vehicles and such other inactive agents well known to the skilled artisan. Vehicles and excipients commonly employed in pharmaceutical preparations include, for example, talc, gum Arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerine and the like. Compositions may be prepared using conventional techniques that may include sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycols mixed with water, Ringer's solution, etc. In one aspect, a coloring agent is added to facilitate in locating and properly placing the composition to the intended treatment site.

Compositions may include a preservative and/or a stabilizer. Non-limiting examples of preservatives include methyl-, ethyl-, propyl-parabens, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride, benzyl alcohol, thimerosal, phenylmercurate salts, chlorhexidine, phenol, 3-cresol, quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, and imidurea.

To control tonicity, the composition can comprise a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride and calcium chloride.

Compositions may include one or more buffers. Typical buffers include: a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffers will typically be included at a concentration in the 5-20 mM range. The pH of a composition will generally be between 5 and 8, and more typically between 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8.

In some embodiments, the composition may include a cryoprotectant agent. Non-limiting examples of cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof.

The genetically engineered microbes may be formulated in a composition for oral administration, for example, as a tablet, capsule, or drink. The genetically engineered microbes may be combined in a liquid carrier. Preferably, the liquid carrier is a moving fluid and the term “liquid carrier” refers to any liquid suitable for ingestion and includes pharmaceutical formulations and foodstuffs such as water, milk, fruit juices, vegetable juices, electrolytic beverages and the like.

The composition can comprise one or more additional substances that can be consumed by the genetically engineered microbe to keep the relevant microbe alive or stimulate its growth. Non-limiting examples of additional substances include mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors and proteins

The composition can be included in an implantable device. Suitable implantable devices contemplated by this invention include encapsulation, scaffolds, grafts, and the like. Such implantable devices can be coated on at least one surface, impregnated, or encapsulated with a composition disclosed and described herein.

Methods of Use

Also provided are methods for killing or inhibiting a pathogenic microbe comprising delivering the genetically engineered microbe as disclosed and described herein to a composition comprising the pathogenic microbe. In some embodiments, the pathogenic microbe is a bacterial pathogen, a viral pathogen, or a fungal pathogen.

In some embodiments, the pathogenic microbe is part of a population of cultured cells (i.e., in vitro). In other embodiments, the pathogenic microbe is part of a population of cells of a subject (i.e., in vivo).

Also provided herein are methods of detecting a state (e.g., virulent or spore) of a pathogenic microbe comprising delivering the genetically engineered microbe as disclosed and described herein to a composition comprising the pathogenic microbe. In some embodiments, the pathogenic microbe is part of a population of cultured cells (i.e., in vitro). In other embodiments, the pathogenic microbe is part of a population of cells of a subject (i.e., in vivo).

Also provided herein are methods of treating or preventing a pathogenic infection in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising genetically engineered microbes as disclosed and described herein.

Subjects treated by the methods disclosed herein include, a simian, a bovine, an equine, a canine, a murine, or a human patient.

In some embodiments, the pathogenic infection is a recurrent pathogenic infection.

In one embodiment, the compositions described herein are useful in treating bacterial infections. Infectious bacteria include, without limitation, Bacillus spp.; Bordetella spp.; Borrelia spp.; Brucella spp.; Burkholderia spp.; Campylobacter spp.; Chlamydia spp.; Chlamydophila spp.; Clostridium spp.; Corynebacterium spp.; Enterococcus spp.; Escherichia spp.; Francisella spp.; Haemophilus spp.; Helicobacter spp.; Legionella spp.; Leptospira spp.; Listeria spp.; Mycobacterium spp.; Mycoplasma spp.; Neisseria spp.; Pseudomonas spp.; Rickettsia spp.; Salmonella spp.; Shigella spp.; Staphylococcus spp.; Streptococcus spp.; Treponema spp.; Vibrio spp.; and Yersinia spp. In one embodiment, the bacterial pathogen is a Clostridium difficile (C. diff). In some embodiments, the bacterial pathogen is an antibiotic-resistant bacterial pathogen. In some embodiments, the bacterial pathogen is a hypervirulent strain.

Non-limiting examples of diseases caused by bacterial pathogens include acute enteritis, anthrax, bacterial meningitis, botulism, brucellosis, cholera, community-acquired respiratory infection, diptheria, dysentery, hemolytic-uremic syndrome, hemorrhagic colitis, leprosy, lyme disease, lymphogranuloma venereum, neumonia, nongonococcal urethritis, sepsis, syphilis (e.g., congential syphilis), tetanus, tuberculosis, typhoid fever, whooping cough, trachoma, inclusion conjunctivitis of the newborn, psittacosis, pseudomembranous colitis, gas gangrene, food poisoning, anaerobic cellulites, nosocomial infections, urinary tract infections, diarrhea, tularemia, upper respiratory tract infections, bronchitis, peptic ulcers, legionnaire's disease, pontiac fever, leptospirosis, listeriosis, tuberculosis, gonorrhea, ophthalmia neonatorum, septic arthritis, meningococcal disease, Waterhouse-Friderichsen syndrome, Pseudomonas infection, rocky mountain spotted fever, typhoid fever type salmonellosis (dysentery, colitis), Salmonellosis with gastroenteritis and/or enterocolitis, bacillary dysentery/Shigellosis, coagulase-positive staphylococcal infections (such as impetigo, acute infective endocarditis, septicemia, necrotizing pneumonia, and toxinoses such as toxic shock syndrome or Staphylococcal food poisoning), cystitis, septicemia, endometritis, otitis media, sinusitis, Streptococcal pharyngitis, scarlet fever, rheumatic fever, erysipelas, puerperal fever, necrotizing fascilitis, bubonic plague and pneumonic plague.

In some embodiments, the pathogen infection is caused by an intestinal and/or gastrointestinal pathogen. Gastrointestinal pathogens include pathogens (e.g., bacteria) that can colonize in the gut of a subject and cause and/or do cause a disease or condition in the subject. Exemplary gastrointestinal pathogens include, but are not limited to Escherichia coli, Clostridium difficile, Clostridium perfringens, Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Enterococcus faecalis (virulent strains of E. faecalis), and Enterococcus faecium.

In one embodiment, the compositions described herein are useful in treating infections by pathogenic viruses. Pathogenic viruses include, without limitation, human papillomavirus, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, Ebola virus, Marburg virus, influenza, respiratory syncytial virus, poxvirus, varicella-zoster virus, and herpes.

Non-limiting examples of viral pathogens include viruses belonging to the following families: Adenoviridae (e.g., adenovirus), Picornaviridae (e.g., coxsackievirus, hepatitis A virus, poliovirus and rhinovirus), Herpesviridae (e.g., herpes simplex type 1, herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, human cytomegalovirus, and human herpesvirus type 8), Hepadnaviridae (e.g., hepatitis B virus), Flaviviridae (e.g., hepatitis C virus, yellow fever virus, dengue virus, and West Nile virus), Retroviridae (e.g., human immunodeficiency virus (HIV)), Orthomyxoviridae (e.g., influenza virus), Paramyxoviridae (e.g., measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus and human metapneumovirus), Papillomaviridae (e.g., papillomavirus), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g., ubella virus) and Parvoviridae (e.g., human bocavirus and parvovirus B19).

Non-limiting examples of diseases caused by viral pathogens include: acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection, gingivostomatitis, tonsillitis, pharyngitis, primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, cytomegalic inclusion disease, Kaposi's sarcoma, Castleman disease, primary effusion lymphoma, AIDS, influenza, Reye syndrome, measles, postinfectious encephalomyelitis, mumps, hyperplastic epithelial lesions, laryngeal papillomas, epidermodysplasia verruciformis, croup, pneumonia, bronchiolitis, common cold, rabies, German measles, congenital rubella, varicella and herpes zoster.

The microbial compositions described herein are useful in treating infections caused by other microbes, including fungus and yeast

Cells can be administered to such an individual by absolute numbers of cells, e.g., said individual can be administered from about 1000 cells/injection to up to about 10 billion cells/injection, such as at about, at least about, or at most about, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³, (and so forth) cells per administration, or any ranges between any two of the numbers, end points inclusive. In other embodiments, cells can be administered to such an individual by relative numbers of cells, e.g., said individual can be administered about 1000 cells to up to about 10 billion cells per kilogram of the individual, such as at about, at least about, or at most about, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³ (and so forth) cells per kilogram of the individual, or any ranges between any two of the numbers, end points inclusive. In some embodiments, between about 1 billion and about 3 billion cells are administered to a patient. In other embodiments, the total dose may be calculated based on m² of body surface area, including 11×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, per m². The average person is 1.6-1.8 m².

In one embodiment, the pathogenic infection is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100% as compared to baseline or a control group receiving a conventional therapy.

In one embodiment, the pathogenic infection is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100% as compared to baseline or a control group receiving a conventional therapy within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 6 months, about 1 year after administering a first dose of the genetically engineered microbes.

In one embodiment, the subject who is administered the genetically engineered microbes experiences and/or reports fewer or less severe side effects on natural gut microbiota as compared to baseline or a control group receiving a conventional therapy. Any method known in the art for assessing natural gut microbiota can be used. Suitable methods include a hydrogen breath test or analyzing a bacterial culture of the small intestine or a stool sample.

In one embodiment, the subject administered the genetically engineered microbe experiences and/or reports fewer or less severe symptoms as compared to baseline or a control group receiving a conventional therapy. Non-limiting examples of symptoms include, fever, pain in abdomen, fatigue, loss of appetite, headache, dry tongue, rice water diarrhea, diarrhea, severe diarrhea with vomiting, slow pulse, cold and clammy skin, dry tongue, severe dehydration, low blood pressure, loss of weight, persistent cough, weakness, occasional blood in sputum, chest pain, burning in urine, painful urination, discharge of pus, yellow or green sputum, pain in chest while coughing, rapid pulse, excessive perspiration, running nose, throat pain, sneezing, itching and burning in nose and eyes, vomiting, muscle pain, rash, itching, or any combination thereof.

Non-limiting examples of conventional therapies for a pathogenic infection include antibacterial drugs (e.g., penicillins, cephalosporins, macrolides, and fluoroquinolones), antiviral drugs (e.g., amantadine, rimantadine, oseltamivir, and zanamivir), and anti-fungal medication (e.g., clotrimazole, econazole nitrate, miconazole, terbinafine, fluconazole, ketoconazole, amphotericin). In some embodiments, the conventional therapy is erythromycin, clindamycin, rifamycin, or any combination thereof.

The composition can be administered by any appropriate route, which will be apparent to the skilled person depending on the disease or condition to be treated. Typical routes of administration include oral, topical, intravenous, intra-arterial, intramuscular, subcutaneous, intracranial, intranasal or intraperitoneal. The composition can be administered by another (e.g., a physician or healthcare provider) or self-administered. In one embodiment, the composition can be administered topically. In another embodiment, the composition can be administered via injection. In a preferred embodiment, the composition can be administrated orally. The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”, “non-parenterally”, and the like, refer to administration of a cell or composition to an individual by a route or mode along the alimentary canal. Examples of “oral” routes of administration of a microbe composition include, without limitation, swallowing liquid or solid forms of a microbe composition from the mouth, administration of a microbe composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a microbe composition, and rectal administration, e.g., using suppositories that release a live microbe described herein to the lower intestinal tract of the alimentary canal.

The composition may also be directly delivered in any manner directly into the gastrointestinal tract. In one embodiment, it is delivered downstream of the stomach directly into the intestines. In another include: (1) placement of a percutaneous endoscopic gastrostomy (PEG) tube and passing a weighted or non-weighted feeding tube into the duodenum or jejunum; (2) surgically placing a direct jejunostomy tube; or, (3) placing a tube directly into the jejunum with a known PEG-like procedure, whereby the jejunum is accessed by stab-piercing the jejunum from outside the abdominal wall.

The dosage and frequency of administration may depend on the type of formulation, route of administration, disease being treated, the amount of cells and any combination therapies, the subject's age, weight, gender, species, other conditions, and the like.

In some embodiments, the composition comprising genetically engineered microbes is administered sequentially or concurrently with an antibiotic. In some embodiments, the genetically engineered microbes are administered after a period of time following administration of an antibiotic. In some embodiments, the subject stops taking the antibiotic prior to administration of the microbes.

EXAMPLES

Example 1: Pathogen-Detecting Sensor Technology

A sensor technology that can turn any specific antibody or binding domain into a cell surface signal will be developed. The antibody or binding domains will be fused to existing cell-surface receptor proteins (i.e., two-component signaling receptors, chemotaxis receptors, quorum sensing circuits) and the fusion proteins will be expressed on the surface of the microbe. FIG. 1. The binding of a pathogen-specific ligand to the antibody or binding domain will either (1) block or disable the binding site of the original receptor, turning the ligand binding into an OFF switch for the chosen signaling circuit or (2) induce a conformational change in the receptor to turn binding into an ON switch. The binding event will then transduce a signal to drive specific transcriptional responses and subsequent expression of specific effectors, for example, a bacteriocin or a protease. Orthogonal binding domains will be designed for specific toxins and surface proteins. Targets include toxins TcdA and TcdB, which are secreted by the virulent form of C. difficile, and spore coat proteins (i.e., BclA glycoproteins and cysteine-rich CdeC). Tying such a sensor to a chemotaxis pathway will allow the engineered microbes to “swim” toward virulent pathogens.

In addition, an infectious pathogen's own sensors can be targeted. Here, a pathogen genome will be mined for sensors of its own environmentally secreted molecules (e.g., quorum sensing receptors). For example, microbes can be engineered to intercept C. difficile siderophores, iron scavenging molecules, to sense the presence of the pathogen and, at the same time, deprive the pathogen of an essential resource.

Example 2: Targeted Countermeasures Against Virulent Pathogen Cells

Microbes will be engineered to produce countermeasures to kill, disable or modify the behavior of the virulent pathogen. Multiple proteins have been identified during a computational screen for native autolysins and C. difficile phage endolysins that may be able to specifically degrade the cell wall of virulent C. difficile to lyse the pathogen. As some of these lysin proteins are native proteins that play an essential role in cell division, it would be difficult for the pathogen to evolve resistance to them. Proteins identified by the screen include those encoded by SEQ ID NOS. 1-40.

Candidate lysin proteins will be screened by high-throughput, cell-free synthesis on a protein array to identify those that show cell-killing activity against C. difficile but not the engineered microbes of the disclosure. Antimicrobial peptides can also be screened for specific activity in killing C. difficile. Effective lysins/peptides can be expressed and secreted upon sensing the presence of the target pathogen. Alternatively, effective lysins/peptides can be produced in an encapsulated form in order to prevent potential toxicity to the host therapeutic agent. To release the encapsulated proteins, the engineered microbe will turn on expression of a bacteriophage lytic protein upon pathogen detection. In addition to the lysins/peptides, encapsulated antitoxins can also be releases to mitigate the effects of the virulent toxins, for example, upon host tissues. The encapsulated shells can be modified with antibodies for targeting to epithelial cells.

Microbes can also be engineered to deliver toxin-specific CRISPR guide RNA (gRNA) to disarm virulent C. difficile. Upon detection of virulent c. difficile, the prophage will be triggered to enter the lytic cycle, cause cell lysis of the engineered microbes and release the phage. The phage can subsequently deliver the CRISPR gRNA to the virulent cell and effectively edit the virulent cell genome to disable the toxin tcdA/B genes, converting the bacterium to a non-virulent form. The presence of non-toxin producing C. difficile can inhibit growth of the virulent form. Nagaro et al., (2013) Antimicrob. Agents Chemother. 57(11):5266-5270. Therefore, by producing more non-virulent C. difficile, it is contemplated that suppression of the virulent form will be stimulated.

Example 3: Targeted Countermeasures Against the Spore State

As a first strategy to selectively kill the highly resistant but dormant C. difficile spores, specific proteases that cleave spore coat proteins will be used. An example of one such target is the cysteine-rich protein CdeC, which has been shown to be essential for assembly of the exosporium layer. Paredes-Sabja et al., (2014) Trends Microbiol. 22(7):406-416. Encapsulated proteases can be delivered, via host microbe lysis, in which the encapsulin shell is targeted to CdeC to provide specificity of cleavage. As a second strategy, microbes can be engineered to metabolize primary bile salts into deoxycholate, which has been shown to induce germination in C. difficile and also inhibit growth of the cells after germination. Sorg et al., (2008) J. Bacteriol. 190 (7):2505-2512. By activating “sleeper” spores, the spores become visible and vulnerable to the arsenal of agents the engineered microbes use to target the virulent state of the pathogen.

Example 4: Engineered Microbes to Dynamically Adapt and Respond to the Pathogen and Host State

Each countermeasure/agent produced by the engineered microbes to target the pathogen can be deployed under the control of an appropriate sensor. For example, the secretion of CRISPR-loaded phages to knockout the pathogen toxin genes can be under control of a toxin sensor, while the secretion of spore coat degrading proteases can be under the control of a spore coat sensor. Each of the feedback loops can be engineered independently (FIG. 2) and in parallel into multiple initial strains of the genetically engineered microbes and dose-response characteristics can be fine-tuned through the choice of suitable transcription factors (TFs), multiplicity of TF binding sites, and autoregulation. Ang et al., (2013) ACS Synth Biol. 2:547-567. It is preferred that responses that involve cell lysis of the engineered microbe be have an all-or-nothing response, so only a fraction of the cells commit suicide. Eldar et al. (2010) Nature 467:167-173. Once each sensor-countermeasure system has been fine-tuned individually, systems can be combined into a single engineered microbe, with some additional regulatory logic to integrate across sensor inputs and response pathways. FIG. 3 and FIG. 4

FIG. 5 shows an example of a dynamically adaptive therapeutic microbe according to an embodiment of the present disclosure that senses both C. difficile spores and virulent cells (i.e., contains a virulent sensor linked to a virulent secretion system and a spore sensor linked to spore section system). The therapeutic microbe/agent has been engineered to detect spore coat proteins. Binding of the spore coat protein to the sensor on the therapeutic agent triggers: (1) signal transductions to cause the therapeutic agent to secrete a compound that induces spores to germinate into a virulent form, for example, a bile salt or (2) release of encapsulated proteases that bind to and kill the C. difficile spores. The therapeutic agent also detects toxins released by the virulent cells. The toxins bind to the virulent sensor on the cell surface of the engineered microbe and activate expression of virulent agents. For example, the engineered microbe can release phages carrying CRISPR gRNA for knockout of toxins whereby the phage infects the C. difficile pathogen and the native CRISPR system disrupts the toxin production to result in a benign C. difficile. In addition, the engineered microbe can release encapsulated drugs, for example lysins. The lysins are released at low pH and kill the pathogenic C. difficile. Finally, the engineered microbe can release encapsulated antitoxins to mitigate the toxin effect on host tissue (e.g., intestinal epithelium cells).

Example 5: Validation of Engineered Microbes

In vitro tests involving a simple two-bacterial system with the engineered microbe and C. difficile, both virulent and spore forms, will be performed. Microscopy and flow cytometry will be utilized to examine how effective the engineered microbes are in neutralizing both forms of C. difficile. Next, combining the engineered microbes in a controlled microbiome, including non-virulent C. difficile and other gut microbes, the changes in the microcosm composition will be measured using next-generation sequencing.

Animal cell lines that are commonly used to model human intestinal disease, for example Caco-2 and HT-29, will be introduced to the engineered microbes to assess the toxicity and efficacy of the microbes in the presence of host cells. Using in vivo animal studies, the engineered microbes will be administered, with and without other gut microbes, after an antibiotic treatment course in mice infected by virulent C. difficile. The health state of the mice will be monitored and the gut microbiome will be analyzed from the fecal microbiota using next generation sequencing.

Finally, in silico computer modeling will be employed to simulate the population dynamics of the microbiome. Data from experimental in vitro and in vivo studies will be incorporated. A conceptual model will be formulated for the native behavior of the gut microbiome during recolonization after broad spectrum antibiotic treatment, with and without the presence of C. difficile, and the tipping point for the onset of C. difficile infection will be identified. Influence of the engineered microbes will then be incorporated into the system. Using these models, the impact of changing given factors (e.g., inoculum size and death rate) to the system will be predicted in order to assess the effectiveness of the engineered microbes and predict the evolutionary behavior of the gut microbiome components of interest. 

What is claimed is:
 1. A genetically engineered microbe comprising a dual pathogen state detection system, the dual pathogen detection system comprising: (a) a first sensor for detection of a virulent form of a pathogen, wherein the first sensor is operatively connected to a virulent secretion system; and (b) a second sensor for detection of a spore form of the pathogen, wherein the second sensor is operatively connected to a spore secretion system.
 2. The microbe of claim 1, wherein the microbe is a bacterium.
 3. The microbe of claim 2, wherein the bacterium is Escherichia coli Nissle 1917 or Lactococcus lactis (L. lactis).
 4. The microbe of claim 1, wherein at least the first sensor or the second sensor detect a protein on the surface of the pathogen.
 5. The microbe of claim 1, wherein the first sensor, the second sensor, or both is a modified cell surface receptor.
 6. The microbe of claim 5, wherein the modified cell surface receptor comprises an antibody, antimicrobial peptide, or fragment thereof.
 7. The microbe of claim 1, wherein the first sensor detects a toxin secreted by the virulent pathogen.
 8. The microbe of claim 7, wherein the toxin is TcdA or TcdB.
 9. The microbe of claim 1, wherein the second sensor detects a cell surface protein on the pathogen.
 10. The microbe of claim 9, wherein the cell surface protein is a cell wall protein or a spore coat protein.
 11. The microbe of claim 10, wherein the spore coat protein is a BclA glycoprotein or a cysteine (CdeC)-rich protein.
 12. The microbe of claim 1, wherein the virulent secretion system produces one or more agents in an encapsulated shell, one or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (gRNA), or any combination thereof.
 13. The microbe of claim 12, wherein the encapsulated agent is a bacteriocin or a modified derivative thereof.
 14. The microbe of claim 13, wherein the bacteriocin is selected from the group consisting of colicin, diffocin, pyocin, and rhuricin
 17. 15. The microbe of claim 13, wherein the encapsulated agent is an autolysin, an endolysin, an antimicrobial peptide, an antitoxin, or any combination thereof.
 16. The microbe of claim 12, wherein the encapsulated shell is modified with an antibody.
 17. The microbe of claim 1, wherein the spore secretion system produces encapsulated agents.
 18. The microbe of claim 17, wherein the encapsulated agents induce germination of spores.
 19. The microbe of claim 17, wherein the encapsulated agents are bile salt or a protease.
 20. A composition comprising an amount of the genetically engineered microbe of claim
 1. 21. A method of inhibiting a pathogenic microbe comprising delivering the genetically engineered microbe of claim 1 to a composition comprising the pathogenic microbe.
 22. A method of detecting a state of a pathogenic microbe comprising delivering the genetically engineered microbe of claim 1 to a composition comprising the pathogenic microbe.
 23. A method of treating or preventing a pathogenic infection in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising genetically engineered microbes of claim
 1. 24. The method of claim 23, wherein the pathogen infection is caused by an intestinal and/or gastrointestinal pathogen.
 25. The method of claim 24, wherein the intestinal pathogen is Clostridium difficile. 